1. Technical Field
The present invention relates to a method for producing an L-amino acid by fermentation, and more specifically to genes which aid in this fermentation. These genes are useful for improving L-amino acid production, for example, for production of L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine, L-tryptophan, and L-glutamic acid.
2. Background Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042 or U.S. Pat. Nos. 4,346,170, 5,661,012 and 6,040,160).
Strains useful in production of L-threonine by fermentation are known, including strains with increased activities of enzymes involved in L-threonine biosynthesis (U.S. Pat. Nos. 5,175,107; 5,661,012; 5,705,371; 5,939,307; EP 0219027), strains resistant to chemicals such as L-threonine and its analogs (WO 01/14525A1, EP 301572 A2, U.S. Pat. No. 5,376,538), strains with target enzymes desensitized to feedback inhibition by the produced L-amino acid or its by-products (U.S. Pat. Nos. 5,175,107; 5,661,012), and strains with inactivated threonine degradation enzymes (U.S. Pat. Nos. 5,939,307; 6,297,031).
The known threonine-producing strain VKPM B-3996 (U.S. Pat. Nos. 5,175,107 and 5,705,371) is presently one of the best known threonine producers. To construct the VKPM B-3996 strain, several mutations and a plasmid, described below, were introduced into the parent strain E. coli K-12 (VKPM B-7). A mutant thrA gene (mutation thrA442) encodes aspartokinase homoserine dehydrogenase I and is resistant to feedback inhibition by threonine. A mutant ilvA gene (mutation ilvA442) encodes threonine deaminase which has a decreased activity, and results in a decreased rate of isoleucine biosynthesis and a leaky phenotype of isoleucine starvation. In bacteria containing the ilvA442 mutation, transcription of the thrABC operon is not repressed by isoleucine, and therefore, these strains are very efficient for threonine production. Inactivation of the tdh gene results in prevention of threonine degradation. The genetic determinant of saccharose assimilation (scrKYABR genes) was transferred to said strain. To increase expression of the genes controlling threonine biosynthesis, the plasmid pVIC40 containing the mutant threonine operon thrA442BC was introduced into the intermediate strain TDH6. The amount of L-threonine which accumulates during fermentation of the strain can be up to 85 g/l.
By optimizing the main biosynthetic pathway of a desired compound, further improvement of L-amino acid producing strains can be accomplished via supplementation of the bacterium with increasing amounts of sugars as a carbon source, for example, glucose. Despite the efficiency of glucose transport by PTS, access to the carbon source in a highly productive strain still may be insufficient.
It is known that the active transport of sugars and other metabolites into bacterial cells is accomplished by several transport systems.
Among these, there are two inducible transport systems for L-arabinose utilization. The low-affinity permease (KM about 0.1 mM) is encoded by the araE gene at min 61.3 and the high-affinity system (KM; 1 to 3 μM) is specified by the araFG operon at min 44.8. The araF gene encodes a periplasmic binding protein (306 amino acids) with chemotactic receptor function and the araG locus encodes at least one inner membrane protein. Both high- and low-affinity transports are under the control of the araC gene product and are thus part of the ara regulon (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C., 1996). Studies in membrane vesicles have shown that L-arabinose permease can transport L-arabinose with low affinity (140-320 μM) and that arabinose transport is coupled with proton transport (Daruwalla, K. R. et al, Biochem. J., 200(3); 611-27 (1981)). L-arabinose permease is a member of the major facilitator superfamily (MFS) of transporters (Griffith, J. K. et al, Curr. Opin. Cell Biol. 4(4); 684-95 (1992)). Imported arabinose is catabolized to xylulose-5-phosphate by a set of enzymes encoded by the araBAD operon, and thence via the pentose phosphate pathway (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C., 1996).
However, there have been no reports to date of using a bacterium of the Enterobacteriaceae family having an enhanced activity of L-arabinose permease for increasing the production of L-amino acids.